2)Charts

• Select the data range you want to apply the chart on

• Go to menu bar and select insert then from it choose chart or press the
chart icon in the formatting tool bar
• A chart wizard is opened for you to select the chart type you want
according to your desire choose the chart type and then click next at the
bottom of the wizard window
• After another click you can write the chart title and name category x and
category y axes
• Also there are tabs beside that one of titles for (axes-gridlines-legend-data
labels-and data tables).
• After pressing next the wizard enable you to save your chart either as an
object in the same sheet or as a new sheet, choose what you want then
click finish.
• HMP Production Whole Genome Shotgun
Data
• The HMP consortium has performed whole genome shotgun sequencing
on samples taken from the digestive tract, mouth, skin, nose, and female
urogenital tract of human subjects to gain insight into the genes and
pathways present in the human microbiome. This data has recently been
deposited into the short read archive at GenBank and the following table
provides links to each sequencing sample. Random identifiers have been
assigned to ensure privacy of the patient. All body site samples derived
from the same patient are linked by a common unique patient identifier.
 HMP
• Traditional microbiology has focused on the study of individual species as isolated
units. However many, if not most, have never been successfully isolated as viable
specimens for analysis, presumably because their growth is dependant upon a
specific microenvironment that has not been, or cannot be, reproduced
experimentally. Among those species that have been isolated, analyses of genetic
makeup, gene expression patterns, and metabolic physiologies have rarely
extended to inter-species interactions or microbe-host interactions. Advances in
DNA sequencing technologies have created a new field of research, called
metagenomics, allowing comprehensive examination of microbial communities,
even those comprised of uncultivable organisms. Instead of examining the
genome of an individual bacterial strain that has been grown in a laboratory, the
metagenomic approach allows analysis of genetic material derived from complete
microbial communities harvested from natural environments. In the HMP, this
method will complement genetic analyses of known isolated strains, providing
unprecedented information about the complexity of human microbial
communities.
 Microbiome Analyses

• There are 10X as many microbial cells as human cells in our bodies. We
are constantly learning more about how changes in the composition of
these communities of organisms (microbiomes) correlate with human
health. Studies have shown that disruptions in our microbiomes may
influence the course of particular disease states. The HMP will expand
upon these studies by performing 16S rRNA and metagenomic sequencing
of samples from a healthy population to address questions such as
whether there is a "core" microbiome at individual body sites and
whether variation in the microbiome can be systematically studied. If you
have questions about this aspect of the HMP, please contact us via the
feedback form in the upper-right hand portion of the screen.
 What is an accession number?

An accession number is label that used to identify a sequence. It is a string of

letters and/or numbers that corresponds to a molecular sequence.

Examples (all for retinol-binding protein, RBP4):

X02775 GenBank genomic DNA sequence

NT_030059 Genomic contig
Rs7079946 dbSNP (single nucleotide polymorphism)
DNA
N91759.1 An expressed sequence tag (1 of 170)
NM_006744 RefSeq DNA sequence (from a transcript)
RNA
NP_007635 RefSeq protein
AAC02945 GenBank protein
Q28369 SwissProt protein
1KT7 Protein Data Bank structure record protein

Page 27
BLAST is…
• Basic Local Alignment Search Tool
• NCBI's sequence similarity search tool
• supports analysis of DNA and protein databases
• 100,000 searches per day

Page 25
Blast
Step 2: Choose the BLAST program
Step 2: Choose the BLAST program
 Activity
• Use the HMP database to find the following:
Acidaminococcus sp.
Gram stain:+
Shape:coccus
Oxygen Req. : Anaerobic
Range: Mesophilic
Protein fasta of conserved hypothetical protein [Acidaminococcus sp. D21] :
MHMLLYVALGGALGSVGRYLVAGSLKGVGGTDFPWHMIAVNTLGCILVGFFVAVLYVKLP
HPRWINLFYWGFIGGFTAFSSFIKEGMHFFLHGEHITGFLYIFLQNMLGMFAAGAAFWLG
KMLL
WGS: assembly fasta
The first gene from ncbi:
1 ttataaagtt ttataaagga cgttgaagac tttcaagagt ttttcgttcc gtctttcggt 61 tgcttcctta tctttcccat tcgagtagtc gtagaagcct
ttcttggtct tcacacccag 121 ttcacccttt tcataatgtt cctttaaaag agtggggatc tcatggctgt catcaaggtc 181 cttcatgagg
taactcgaga catggtagaa cgtgtcgatc ccgccaaaat ccatggtttc 241 gagcggtcca atgcaggccc agcggaaagc aagtccatat
ttcataacag cgtcaatatc 301 ttcagcagaa acaacacctt ttttcaccaa agacagggct tcccgcacga cagccagctg 361
gatgcggttt gcagcaaaac ccaggacatc cttattgaca atgaccggtt tctttccaat 421 ggtgcgggca aggtcccgaa cggcctcagc
cacgtcttgg caggtttcgt catttttaat 481 gatttcaata aggagaataa gcgtcggcgg attgaaccag tgcatcccta aaaagcgttc 541
cctgtgcgtc acaaattggg ccagggcatt gatggaaaga cctgatgtat ttgtcgcaag 601 gatcatgtcc gcatcggcca tcttgcagaa
agattcatag aacccctctt taatggccat 661 gtcttccgtc acgttttcaa tgacaatatc gagatgggcg atgtcctcca gattggtcgt 721
atagtggatc ttgtcgcggc tcgtttcact gatgagggtc tttgcacgct caagcgtagg 781 ctttctatgg ttccaaaggg tcacatcaaa
accataggaa gcaaagatat ccgccatgga 841 atagcccatc gtaccagcgc cggcaatgcc gattcgtttg atctccataa tcaaggctcc
901 tttcaagatc at
Then make blast of the obtained gene against database
• Use the cmr to find the following:
Staphylococcus aureus
how many strains are there ?14,
select the first one : Staphylococcus aureus
MW2,
taxon ID:196620
no. of protein coding genes:2632
G+C %: 32.82%
find the sequencing center for it: Juntendo Univ NITE 
• bacillus subtilis
how many strains are there ? 1
select the first one : Bacillus subtilis 168
taxon ID: 224308
no. of protein coding genes: 4245
A+T %: 56.48%
find the sequencing center for it: Japanese
Consortium European Consortium 
Sequence Length: 4215606 bp
16s rRNA
The Biomarker 16S rDNA - identify and
classify organisms by
gene sequence
16S rDNA variations.

rRNA (functional molecule)

LSU

SSU
 C. perfringens probe set identified in
EPA sample 22 (N.Y. Spring)

C.AURANTIBUTYRICUM
CFB
C.THERMOBUTYRICUM_SUBGROUP C. BUTYRICUM
Cyan High G+C C.ALGIDICARNIS
Bacteria Proteo C.BOTULINUM_SUBGROUP C.CADAVERIS
Bacil-Strep
Gram + C.PERFRINGENS
C.BARATI_SUBGROUP
Clostridium

27 1492

16S rDNA
420 469
...CGTAAAGCTCTGTCTTTGGGGAAGATAATGACGGTACCCAAGGAGGAAGCCACGGCTAACT... C. perf. str.CPN50
5 6 7 8
................................................................... C. perf. resistant
................................................................... Clostridium sp. AB&J
................................................................... clone p-4636-2Wa2
................................................................... C. perf. A
................................................................... C. perf rrnA
................................................................... C. perf rrnE
.................................T................................. C. perf rrnD
................................................................... C. perf rrnC
................................................................... C. perf rrnB
................................................................... C. perf rrnF
................................................................... C. perf rrnG
................................................................... C. perf str.13a
................................................................... C. perf str.13b
................................................................... C. perf rrnH
................................................................... C. perf rrnI
...................................................................
...................................................................
C. perf rrnJ
clone OI1612 Ave Diff =1891
................................................................... C. perf. B

Probe Properties:
................................................................... Swine manure 37-3
................................................................... Swine manure 37-4

TAAAGCTCTGTCTTTGGGGAAGATA
AAAGCTCTGTCTTTGGGGAAGATAA
AAGCTCTGTCTTTGGGGAAGATAAT
tacccaaggaggaagccacggctaa
25mer exits in 90% of the taxon’s seqs
AGCTCTGTCTTTGGGGAAGATAATG

Probes 5 - 8 Internal 21mer exists only in one taxon.

 Why the ribosomal RNA gene?

 High content of information

– 500 bp sequence with 4 different bases
 4500 = 1 x 10301 variants
– 15 biochemical tests with “yes/no” result
 215 = 3 x 104 variants

 16S rDNA has become the Standard for

Taxonomic Classification
– Bergey’s Manual of Systematic Bacteriology
Q: What is the Expect (E) value?
The Expect value (E) is a parameter that describes the number of hits one can
"expect" to see by chance when searching a database of a particular size. It
decreases exponentially as the Score (S) of the match increases. Essentially, the
E value describes the random background noise. For example, an E value of 1
assigned to a hit can be interpreted as meaning that in a database of the current
size one might expect to see 1 match with a similar score simply by chance.
The lower the E-value, or the closer it is to zero, the more "significant" the match
is. However, keep in mind that virtually identical short alignments have relatively
high E values. This is because the calculation of the E value takes into account
the length of the query sequence. These high E values make sense because
shorter sequences have a higher probability of occurring in the database purely by
chance. For more details please see the calculations in the BLAST Course.
The Expect value can also be used as a convenient way to create a significance
threshold for reporting results. You can change the Expect value threshold on
most BLAST search pages. When the Expect value is increased from the default
value of 10, a larger list with more low-scoring hits can be reported.
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?
CMD=Web&PAGE_TYPE=BlastDocs&DO
C_TYPE=FAQ#expect